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• W2121722333 abstract "NLRP12 is a member of the intracellular Nod-like receptor (NLR) family that has been suggested to downregulate the production of inflammatory cytokines, but its physiological role in regulating inflammation has not been characterized. We analyzed mice deficient in Nlrp12 to study its role in inflammatory diseases such as colitis and colorectal tumorigenesis. We show that Nlrp12-deficient mice are highly susceptible to colon inflammation and tumorigenesis, which is associated with increased production of inflammatory cytokines, chemokines, and tumorigenic factors. Enhanced colon inflammation and colorectal tumor development in Nlrp12-deficient mice are due to a failure to dampen NF-κB and ERK activation in macrophages. These results reveal a critical role for NLRP12 in maintaining intestinal homeostasis and providing protection against colorectal tumorigenesis. NLRP12 is a member of the intracellular Nod-like receptor (NLR) family that has been suggested to downregulate the production of inflammatory cytokines, but its physiological role in regulating inflammation has not been characterized. We analyzed mice deficient in Nlrp12 to study its role in inflammatory diseases such as colitis and colorectal tumorigenesis. We show that Nlrp12-deficient mice are highly susceptible to colon inflammation and tumorigenesis, which is associated with increased production of inflammatory cytokines, chemokines, and tumorigenic factors. Enhanced colon inflammation and colorectal tumor development in Nlrp12-deficient mice are due to a failure to dampen NF-κB and ERK activation in macrophages. These results reveal a critical role for NLRP12 in maintaining intestinal homeostasis and providing protection against colorectal tumorigenesis. NLRP12 dampens inflammation and tumorigenesis in the colon NLRP12 regulates cytokine and chemokine production, and epithelial proliferation NLRP12 negatively regulates NF-κB and ERK activation in the macrophages NLRP12 activity in myeloid compartment is essential for colonic homeostasis Colorectal cancer is the third most common form of cancer and the second leading cause of cancer-related death in developed countries. Chronic inflammation shapes the tumorigenic micro-environment in the gut by inducing cytokines, chemokines, and other factors through NF-κB, ERK, and STAT3 signaling. In this study, we showed that the NOD-like receptor family member NLRP12 plays a critical role in downregulating these tumor-inducing signaling pathways. Given the importance of anti-inflammatory signals in maintaining colonic homeostasis, these results reveal a regulatory mechanism controlling inflammation and tumorigenesis in the gut, and may help identify new therapeutic approaches to control inflammatory bowel diseases. Inflammation is generally considered to be a host protective response against infection and injury (Medzhitov, 2008Medzhitov R. Origin and physiological roles of inflammation.Nature. 2008; 454: 428-435Crossref PubMed Scopus (3929) Google Scholar). However, uncontrolled inflammation is a major risk factor for the development of cancer (Grivennikov et al., 2010Grivennikov S.I. Greten F.R. Karin M. Immunity, inflammation, and cancer.Cell. 2010; 140: 883-899Abstract Full Text Full Text PDF PubMed Scopus (7487) Google Scholar). Colorectal cancer is the third most common form of cancer and the second leading cause of cancer-related death in developed countries (Eaden et al., 2001Eaden J.A. Abrams K.R. Mayberry J.F. The risk of colorectal cancer in ulcerative colitis: a meta-analysis.Gut. 2001; 48: 526-535Crossref PubMed Scopus (2299) Google Scholar, Ekbom et al., 1990Ekbom A. Helmick C. Zack M. Adami H.O. Ulcerative colitis and colorectal cancer. A population-based study.N. Engl. J. Med. 1990; 323: 1228-1233Crossref PubMed Scopus (1563) Google Scholar, Itzkowitz and Yio, 2004Itzkowitz S.H. Yio X. Inflammation and cancer IV. Colorectal cancer in inflammatory bowel disease: the role of inflammation.Am. J. Physiol. Gastrointest. Liver Physiol. 2004; 287: G7-G17Crossref PubMed Scopus (1033) Google Scholar). Notably, patients with inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis are at increased risk for the development of colorectal cancer (Fiocchi, 1998Fiocchi C. Inflammatory bowel disease: etiology and pathogenesis.Gastroenterology. 1998; 115: 182-205Abstract Full Text Full Text PDF PubMed Scopus (1864) Google Scholar). Although the precise molecular mechanism of IBD-related colorectal tumor formation is incompletely understood, it is widely viewed that cytokines, chemokines, matrix degrading enzymes, and growth factors produced during chronic inflammation in IBD patients contribute to mutagenic transformation of colonic epithelial cells into neoplastic cells (Grivennikov et al., 2010Grivennikov S.I. Greten F.R. Karin M. Immunity, inflammation, and cancer.Cell. 2010; 140: 883-899Abstract Full Text Full Text PDF PubMed Scopus (7487) Google Scholar). Chronic inflammatory diseases of the gut are initiated by the aberrant interaction of the host immune system with commensal microflora (Goyette et al., 2007Goyette P. Labbé C. Trinh T.T. Xavier R.J. Rioux J.D. Molecular pathogenesis of inflammatory bowel disease: genotypes, phenotypes and personalized medicine.Ann. Med. 2007; 39: 177-199Crossref PubMed Scopus (81) Google Scholar, Zaki et al., 2011Zaki M.H. Lamkanfi M. Kanneganti T.D. The Nlrp3 inflammasome: contributions to intestinal homeostasis.Trends Immunol. 2011; 32: 171-179Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Innate immune receptors such as Toll-like receptors (TLR) at the surface of epithelial cells and immune cells initiate this inflammatory process by activating the downstream transcription factor NF-κB, which is a central mediator of proinflammatory cytokine and chemokine production. However, a tight regulation of NF-κB signaling is essential to maintaining a beneficial level of homeostatic interactions with the gut microflora. Therefore, deregulated NF-κB signaling may represent a key mechanism contributing to gut inflammation, colitis, and colorectal tumorigenesis (Leu et al., 2003Leu C.M. Wong F.H. Chang C. Huang S.F. Hu C.P. Interleukin-6 acts as an antiapoptotic factor in human esophageal carcinoma cells through the activation of both STAT3 and mitogen-activated protein kinase pathways.Oncogene. 2003; 22: 7809-7818Crossref PubMed Scopus (142) Google Scholar, van Vliet et al., 2005van Vliet C. Bukczynska P.E. Puryer M.A. Sadek C.M. Shields B.J. Tremblay M.L. Tiganis T. Selective regulation of tumor necrosis factor-induced Erk signaling by Src family kinases and the T cell protein tyrosine phosphatase.Nat. Immunol. 2005; 6: 253-260Crossref PubMed Scopus (140) Google Scholar, Yu et al., 2009Yu H. Pardoll D. Jove R. STATs in cancer inflammation and immunity: a leading role for STAT3.Nat. Rev. Cancer. 2009; 9: 798-809Crossref PubMed Scopus (3106) Google Scholar). Recent studies demonstrated a key role for molecules that negatively regulate NF-κB activation in maintenance of gut homeostasis. For instance, enterocyte-specific deletion of the NF-κB regulator A20 rendered mice hypersusceptible to colitis and colorectal tumorigenesis as a consequence of uncontrolled production of NF-κB-dependent proinflammatory cytokines (Lee et al., 2000Lee E.G. Boone D.L. Chai S. Libby S.L. Chien M. Lodolce J.P. Ma A. Failure to regulate TNF-induced NF-kappaB and cell death responses in A20-deficient mice.Science. 2000; 289: 2350-2354Crossref PubMed Scopus (1192) Google Scholar, Vereecke et al., 2010Vereecke L. Sze M. McGuire C. Rogiers B. Chu Y. Schmidt-Supprian M. Pasparakis M. Beyaert R. van Loo G. Enterocyte-specific A20 deficiency sensitizes to tumor necrosis factor-induced toxicity and experimental colitis.J. Exp. Med. 2010; 207: 1513-1523Crossref PubMed Scopus (228) Google Scholar). Similarly, mice deficient in TIR8/SIGGIR, a molecule that negatively regulates Toll-like receptor (TLR)- and interleukin (IL)-1 receptor-mediated NF-κB signaling, suffered from increased susceptibility to colitis and colorectal tumorigenesis (Garlanda et al., 2004Garlanda C. Riva F. Polentarutti N. Buracchi C. Sironi M. De Bortoli M. Muzio M. Bergottini R. Scanziani E. Vecchi A. et al.Intestinal inflammation in mice deficient in Tir8, an inhibitory member of the IL-1 receptor family.Proc. Natl. Acad. Sci. USA. 2004; 101: 3522-3526Crossref PubMed Scopus (231) Google Scholar, Xiao et al., 2010Xiao H. Yin W. Khan M.A. Gulen M.F. Zhou H. Sham H.P. Jacobson K. Vallance B.A. Li X. Loss of single immunoglobulin interlukin-1 receptor-related molecule leads to enhanced colonic polyposis in Apc(min) mice.Gastroenterology. 2010; 139: 574-585Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In addition to TLRs, the immune system makes use of a limited set of germ-line encoded pattern recognition receptors (PRRs) to induce the production of inflammatory cytokines in response to microbial components (Kawai and Akira, 2007Kawai T. Akira S. Signaling to NF-kappaB by Toll-like receptors.Trends Mol. Med. 2007; 13: 460-469Abstract Full Text Full Text PDF PubMed Scopus (1646) Google Scholar). This includes C-type lectin receptors (CLRs), RIG-I-like receptors (RLRs), HIN-200 proteins and nucleotide binding, and oligomerization domain-like receptors (NLRs) (Inohara et al., 2005Inohara Chamaillard McDonald C. Nuñez G. NOD-LRR proteins: role in host-microbial interactions and inflammatory disease.Annu. Rev. Biochem. 2005; 74: 355-383Crossref PubMed Scopus (811) Google Scholar, Kanneganti et al., 2006Kanneganti T.D. Ozören N. Body-Malapel M. Amer A. Park J.H. Franchi L. Whitfield J. Barchet W. Colonna M. Vandenabeele P. et al.Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3.Nature. 2006; 440: 233-236Crossref PubMed Scopus (900) Google Scholar, Kanneganti et al., 2007Kanneganti T.D. Lamkanfi M. Núñez G. Intracellular NOD-like receptors in host defense and disease.Immunity. 2007; 27: 549-559Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar). NLR proteins represent platform proteins that are characterized by the presence of a conserved nucleotide binding and oligomerization domain (referred to as NBD; NOD or NACHT domain) and are located in intracellular compartments (Kanneganti et al., 2007Kanneganti T.D. Lamkanfi M. Núñez G. Intracellular NOD-like receptors in host defense and disease.Immunity. 2007; 27: 549-559Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar). NLRs are implicated in a multitude of innate immune signaling pathways ranging from the regulation of MAP kinase and NF-κB signaling pathways by NOD1 and NOD2, over modulation of MHC class II genes by CIITA, to the assembly of caspase-1-activating protein complexes named “inflammasomes” by the NLR proteins NLRP1, NLRP3, and NLRC4 (Kanneganti et al., 2007Kanneganti T.D. Lamkanfi M. Núñez G. Intracellular NOD-like receptors in host defense and disease.Immunity. 2007; 27: 549-559Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar). Unlike the above-mentioned NLRs, the in vivo role of the NLR protein NLRP12 is not clear. Notably, polymorphisms in the gene encoding NLRP12 have been linked with increased susceptibility to periodic fever syndromes and atopic dermatitis (Arthur et al., 2010Arthur J.C. Lich J.D. Ye Z. Allen I.C. Gris D. Wilson J.E. Schneider M. Roney K.E. O'Connor B.P. Moore C.B. et al.Cutting edge: NLRP12 controls dendritic and myeloid cell migration to affect contact hypersensitivity.J. Immunol. 2010; 185: 4515-4519Crossref PubMed Scopus (115) Google Scholar, Borghini et al., 2011Borghini S. Tassi S. Chiesa S. Caroli F. Carta S. Caorsi R. Fiore M. Delfino L. Lasiglie D. Ferraris C. et al.Clinical presentation and pathogenesis of cold-induced autoinflammatory disease in a family with recurrence of a NLRP12 mutation.Arthritis Rheum. 2011; 63: 830-839Crossref PubMed Scopus (141) Google Scholar, Jéru et al., 2008Jéru I. Duquesnoy P. Fernandes-Alnemri T. Cochet E. Yu J.W. Lackmy-Port-Lis M. Grimprel E. Landman-Parker J. Hentgen V. Marlin S. et al.Mutations in NALP12 cause hereditary periodic fever syndromes.Proc. Natl. Acad. Sci. USA. 2008; 105: 1614-1619Crossref PubMed Scopus (284) Google Scholar, Macaluso et al., 2007Macaluso F. Nothnagel M. Parwez Q. Petrasch-Parwez E. Bechara F.G. Epplen J.T. Hoffjan S. Polymorphisms in NACHT-LRR (NLR) genes in atopic dermatitis.Exp. Dermatol. 2007; 16: 692-698Crossref PubMed Scopus (91) Google Scholar). NLRP12 was initially shown to regulate NF-κB and caspase-1 activation (Wang et al., 2002Wang L. Manji G.A. Grenier J.M. Al-Garawi A. Merriam S. Lora J.M. Geddes B.J. Briskin M. DiStefano P.S. Bertin J. PYPAF7, a novel PYRIN-containing Apaf1-like protein that regulates activation of NF-kappa B and caspase-1-dependent cytokine processing.J. Biol. Chem. 2002; 277: 29874-29880Crossref PubMed Scopus (303) Google Scholar). Moreover, NLRP12 was recently suggested to negatively regulate canonical and noncanonical NF-κB signaling in vitro by targeting the kinases IRAK1 and NIK for proteasomal degradation (Arthur et al., 2007Arthur J.C. Lich J.D. Aziz R.K. Kotb M. Ting J.P. Heat shock protein 90 associates with monarch-1 and regulates its ability to promote degradation of NF-kappaB-inducing kinase.J. Immunol. 2007; 179: 6291-6296PubMed Google Scholar, Lich et al., 2007Lich J.D. Williams K.L. Moore C.B. Arthur J.C. Davis B.K. Taxman D.J. Ting J.P. Monarch-1 suppresses non-canonical NF-kappaB activation and p52-dependent chemokine expression in monocytes.J. Immunol. 2007; 178: 1256-1260PubMed Google Scholar, Williams et al., 2005Williams K.L. Lich J.D. Duncan J.A. Reed W. Rallabhandi P. Moore C. Kurtz S. Coffield V.M. Accavitti-Loper M.A. Su L. et al.The CATERPILLER protein monarch-1 is an antagonist of toll-like receptor-, tumor necrosis factor alpha-, and Mycobacterium tuberculosis-induced pro-inflammatory signals.J. Biol. Chem. 2005; 280: 39914-39924Crossref PubMed Scopus (168) Google Scholar). However, NLRP12 missense mutations in periodic fever syndrome patients were recently linked to increased caspase-1 activation rather than to inhibition of NF-κB signaling (Borghini et al., 2011Borghini S. Tassi S. Chiesa S. Caroli F. Carta S. Caorsi R. Fiore M. Delfino L. Lasiglie D. Ferraris C. et al.Clinical presentation and pathogenesis of cold-induced autoinflammatory disease in a family with recurrence of a NLRP12 mutation.Arthritis Rheum. 2011; 63: 830-839Crossref PubMed Scopus (141) Google Scholar, Jéru et al., 2010Jéru I. Marlin S. Le Borgne G. Cochet E. Normand S. Duquesnoy P. Dastot-Le Moal F. Cuisset L. Hentgen V. Fernandes Alnemri T. et al.Functional consequences of a germline mutation in the leucine-rich repeat domain of NLRP3 identified in an atypical autoinflammatory disorder.Arthritis Rheum. 2010; 62: 1176-1185Crossref PubMed Scopus (23) Google Scholar). Therefore, the physiological relevance of NLRP12-mediated regulation of NF-κB pathways remains to be defined. In this study we focused on determining the physiological role of NLRP12 in regulating inflammation in a mouse model of colitis and colorectal tumorigenesis. Mouse NLRP12 shares with other NLRs a structural composition that exists of an amino-terminal Pyrin motif, followed by a central nucleotide-binding domain (NBD) and a C-terminal leucine rich repeat domain (see Figure S1A available online). The product is encoded on mouse chromosome 7 and contains 10 exons spanning a region of 28.3 kb. We initially investigated the expression pattern of murine Nlrp12 transcripts in a variety of primary immune cell populations. Cells with the highest expression levels of Nlrp12 mRNA were neutrophils and T cells, followed by dendritic cells and macrophages, respectively (Figure 1A ). Recently, it was reported that NLRP12 is expressed in the colon tissue (Lech et al., 2010Lech M. Avila-Ferrufino A. Skuginna V. Susanti H.E. Anders H.J. Quantitative expression of RIG-like helicase, NOD-like receptor and inflammasome-related mRNAs in humans and mice.Int. Immunol. 2010; 22: 717-728Crossref PubMed Scopus (123) Google Scholar). We further verified the expression of Nlrp12 in different parts of the gastrointestinal tract and lymphoid organs. Nlrp12 was found to be expressed in the small intestine, caecum, colon, spleen, liver, and mesenteric lymph nodes (MLN) (Figure 1B). To define the role of NLRP12 in regulating inflammatory responses in the gut, Nlrp12-deficient mice were generated by homologous recombination. To this aim, exon II encoding the Pyrin domain of Nlrp12—that is required for the recruitment of downstream effectors and functions of the protein—was replaced with a neomycin selection cassette in the targeting construct (Figures S1B and S1C). Positive embryonic stem (ES) cells were used to generate chimeric mice and Nlrp12−/− mice were backcrossed to the C57BL/6 genetic background for 10 generations. Nlrp12−/− mice were fertile and appeared healthy when housed in a specific pathogen-free environment. To define the role of NLRP12 in colitis-induced inflammation, Nlrp12−/− mice were fed 3% DSS in drinking water for 5 days and susceptibility was monitored by measuring body weight, assessing stool consistency and rectal bleeding, and measuring colon length during both the acute (day 5) and recovery (days 9–20) stages of disease. Notably, disease progression and clinical scores of wild-type and Nlrp12-deficient mice were not statistically different during the acute phase of disease. However, wild-type mice started to recover once DSS was omitted from the drinking water, whereas Nlrp12-deficient mice suffered from continued body weight loss (Figure 1C), diarrhea, and rectal bleeding (Figure 1D). This inflammatory phenotype was further evidenced by the gross appearance of the colon. During the acute stage of colitis (at day 5), colons of wild-type and Nlrp12-deficient mice appeared similar (data not shown). At day 9, however, colons of Nlrp12-deficient mice were significantly shorter than those of wild-type mice (Figures 1E and 1F). To determine whether recovery in Nlrp12−/− mice was simply delayed, or whether NLRP12-deficiency prevented healing responses at later time points as well, colon length, and weight of Nlrp12−/− mice were analyzed at day 20 after DSS-induced colitis. Notably, colons of Nlrp12−/− mice were significantly shorter and weighted more than those of wild-type mice (Figures S1D and S1E), suggesting that Nlrp12−/− mice continued to suffer from colon inflammation 2 weeks after DSS administration was stopped. Consistent with an inflammatory phenotype, MLN and spleens of DSS-fed Nlrp12−/− mice were found to be significantly heavier and enlarged at day 20 compared to those of wild-type mice (Figures S1F and S1G). To obtain further evidence of sustained inflammation in Nlrp12-deficient mice, colon tissue was analyzed histologically at days 5, 9, and 20 following DSS administration. Consistent with the clinical parameters discussed above, colons of Nlrp12-deficient mice contained markedly more infiltrating inflammatory cells, and displayed significantly more ulceration and hyperplasia during the recovery phase of the disease (at days 9 and 20), but not at early stages (Figures 1G and 1H). In agreement, colon tissue of Nlrp12-deficient mice contained significantly higher levels of proinflammatory cytokines than wild-type mice at day 9 (Figure S1H), but not at day 5 post-DSS administration (data not shown). Together, these results indicate that NLRP12 plays a critical role in resolving the inflammatory response following DSS-induced injury of the colonic epithelium. The observation that Nlrp12-deficient mice suffered from sustained gut inflammation upon DSS-treatment, prompted us to investigate the role of Nlrp12 in colitis-associated tumorigenesis. To this aim, we induced colon tumorigenesis by injecting a single dose of the DNA-methylating agent azoxymethane (AOM), which was followed by three cycles of 3% DSS-administration (Figure 2A ). Changes in body weight were monitored daily throughout the study duration and colonic tumor burden was determined 12 weeks after AOM/DSS treatment. Nlrp12-deficient mice lost significantly more body weight relative to wild-type mice (Figure 2B) and showed increased rectal bleeding (Figure S2A). Consistently, Nlrp12-deficient mice had significantly higher tumor burdens in the colon, although tumor size was not significantly different (Figures 2C–2E). Tumors in wild-type mice were mostly contained within the colorectal and distal areas of the colon, whereas tumors in Nlrp12-deficient mice were commonly found throughout the entire colonic tract (Figures S2B and S2C). Increased tumor burdens in Nlrp12-deficient mice were associated with more inflammation and hyperplasia (Figures 2F and 2G), and a higher incidence of dysplasia (Figure 2H). Histological examination of tumors and adenomatous polyps showed that all Nlrp12-deficient mice included in the study developed high-grade dysplasia, of which ∼30% were classified as adenocarcinoma (Figures 2H and 2I). By contrast, only 20% of the wild-type cohort displayed high-grade dysplasia in the colon, and adenocarcinoma development was not evident in this group (Figure 2I). Collectively, these results indicate a critical role for NLRP12 in protection against colitis-associated tumorigenesis. The splenomegaly of DSS-fed Nlrp12-deficient mice (Figure S1G) was also apparent in AOM/DSS-administered animals (Figures S3A and S3B). We therefore hypothesized that NLRP12 may protect from colitis-associated tumorigenesis by dampening immune cell activation and inflammatory responses in response to DSS-treatment. To characterize this possibility in additional detail, we carefully examined the histopathological changes that occur during early stages of tumor induction (at day 15 after AOM injection). In line with our hypothesis, histological analysis of colon sections revealed markedly more tissue damage, mucosal edema, inflammation, and hyperplasia in Nlrp12-deficient mice than in wild-type mice (Figures 3A and 3B). Semiquantitative scores for colon inflammation, ulceration, and hyperplasia were all significantly higher in Nlrp12-deficient mice (Figure S3C). Moreover, Nlrp12-deficient colons showed increased infiltration of macrophages, PMNs, and T cells (Figure 3C). Hyperinfiltration of immune cells in Nlrp12−/− mice was not confined to inflamed sections of the colon, but extended to the entire colon as evidenced by an increased F4/80-staining in relatively noninflamed parts of the Nlrp12−/− colon (Figure 3D). To further characterize the immune cell types associated with the induction of hyperinflammatory responses in the colon of Nlrp12−/− mice, myeloid cells present in the colonic lamina propria were isolated at different stages of colitis and analyzed by flow cytometry. During early stages of colitis (at day 5), neutrophils were the most prevalent cell type found in the lamina propria of both wild-type and Nlrp12−/− mice, but their number was nearly doubled in Nlrp12-deficient colons (Figure 3E). Notably, at later stages of colitis (days 9 and 20), cell counts of all analyzed myeloid cell types (CD11b+, F4/80+, CD11c+, Gr-1+) in Nlrp12−/− mouse colons were significantly higher than in wild-type colons (Figures 3F and 3G). At day 20, a similarly dramatic increase in infiltration of myeloid cell populations was evident in the mesenteric lymph node (MLN) and spleen of Nlrp12−/− mice, although myeloid cell counts in these tissues were comparable to those of wild-type mice at earlier time points (Figure 3G; Figure S3D). Notably, number and frequency of myeloid cell populations in untreated control mice of wild-type and Nlrp12−/− background were not different (data not shown). In addition to being more prevalent, a larger number of CD11b+ myeloid cells that were collected from the spleen and MLN of Nlrp12−/− mice at day 20 produced IL-6 and TNF-α in response to LPS and following PMA plus ionomycin stimulation (Figure 4A ). Moreover, the mean fluorescent intensity (MFI) for intracellular expression for IL-6 and TNF-α was significantly higher for Nlrp12-deficient CD11b+ cells than for wild-type cells, indicating that Nlrp12−/− myeloid cells produced higher levels of these proinflammatory cytokines (Figure 4B). Thus, together these results indicate that NLRP12 plays a critical role in dampening the inflammatory response in myeloid cells and during DSS-induced colitis. Consistent with the enhanced infiltration and hyperactivation of myeloid cells in the absence of NLRP12, the production of proinflammatory cytokines such as IL-1β, IL-6, TNF-α, IL-17, and IL-15 were all found to be elevated in the colon of Nlrp12−/− mice relative to the levels found in wild-type mice (Figure 4C and data not shown). Similarly, colons of Nlrp12-deficient mice contained higher levels of the chemokines G-CSF, eotaxin, KC, IP-10, MIP-1α, MIP-1β, and MIP2 (Figure 4C and data not shown). Given the association of higher tumor burdens with enhanced production of proinflammatory cytokines and chemokines in Nlrp12−/− mice, we hypothesized that unlike most NLRs, NLRP12 may operate as a negative regulator of inflammatory signaling pathways. Such mechanism may also explain its antitumor function because increased cytokine and chemokine production, along with tumorigenic growth factors, may create a microenvironment that promotes unwarranted cell proliferation and adenomatous polyp development in Nlrp12−/− mice. To understand the nature of the tumorigenic signals that are deregulated in Nlrp12−/− mice, we first studied apoptosis induction in colon tissue of AOM/DSS-treated mice. However, mRNA and protein expression of the antiapoptotic protein Bcl-XL as well as the number of TUNEL-positive cells in colon tissue of wild-type and Nlrp12−/− mice were comparable (Figure S4). We next analyzed the expression of multiple cytokines and tumorigenic factors such as cycloxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS /NOS2) in colon tissue because these factors often drive tumorigenesis. The transcript level of proinflammatory cytokines such as IL-6 and TNF-α and the chemokine MIP2 were markedly more induced in Nlrp12−/− mice relative to the levels in wild-type colon (Figure 5A ). Unlike IL-6 and TNF-α, the levels of the tumor-suppressing cytokine IFN-γ and its effector IFN-γ-dependent NOS2 transcripts were not significantly changed (Figure 5A). In contrast, we measured 3-fold higher mRNA levels of the prostaglandin-synthesizing enzyme COX2, which has previously been linked to colon carcinogenesis (Buchanan and DuBois, 2006Buchanan F.G. DuBois R.N. Connecting COX-2 and Wnt in cancer.Cancer Cell. 2006; 9: 6-8Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, Shiff and Rigas, 1999Shiff S.J. Rigas B. The role of cyclooxygenase inhibition in the antineoplastic effects of nonsteroidal antiinflammatory drugs (NSAIDs).J. Exp. Med. 1999; 190: 445-450Crossref PubMed Scopus (143) Google Scholar). Moreover, elevated COX2 and MIP2 expression was also evident in colonic tumors of Nlrp12-deficient mice (Figure 5B). The expression of tumorigenic and proinflammatory genes is modulated by signal transduction pathways mediated by NF-κB, MAPK, STAT, and AKT proteins. To understand whether these pathways and molecules were deregulated in the absence of Nlrp12, we examined activation of NF-κB, MAPK, and STAT signaling pathways by western blotting. Indeed, significantly higher activation levels of NF-κB, ERK, and STAT3 were observed in Nlrp12-deficient colon tissue at day 15 after AOM injection (day 10 after DSS) relative to the levels found in wild-type mice (Figures 5C and 5D). Hyperactivation of these inflammatory pathways is associated with an increased proliferation of epithelial cells in hyperplastic colon regions of AOM/DSS treated Nlrp12-deficient mice (Figures 5E and 5F). By contrast, untransformed colon tissue and unaffected colon regions of both Nlrp12−/− and wild-type mice displayed similar proliferation levels as measured by BrdU staining (data not shown). Notably, hyperplastic colonic tissue in Nlrp12-deficient mice was surrounded by a massive infiltration of macrophages (Figure 5G), suggesting that myeloid cells may provide signals that promote tumorigenesis in the absence of NLRP12 signaling. To determine the cell populations that contribute to NLRP12-mediated protection against tumorigenesis, we generated 4 groups of Nlrp12 bone marrow chimeras (Figure 6A ). Eight weeks after bone marrow reconstitution, mice were subjected to tumor induction using AOM plus DSS. NLRP12 deficiency in either compartment led to increased body weight loss compared to wild-type mice (Figure 6A), suggesting that NLRP12 may contribute to protection against colitis in both immune and nonimmune cells. However, the body weight of Nlrp12-deficient mice with wild-type hematopoietic cells later recovered to level similar to those of wild-type mice, whereas mice lacking NLRP12 in the hematopoietic compartment failed to do so (Figure 6A). This suggests that NLRP12 signaling in immune cells is critical to controlling colitis, whereas its role in epithelial cells may be redundant. Consistently, the groups lacking Nlrp12 in the hematopoietic compartment had significantly higher tumor burdens and shortened colons (Figures 6B and 6C). On the other hand, no significant differences in tumor burdens and colon length were observed between wild-type and Nlrp12-deficient mice having wild-type bone marrow cells (Figures 6B and 6C). In agreement, colon tissue of chimera groups with Nlrp12-deficient immune cells that was collected at day 15 after AOM injection (day 10 after DSS) showed increased NF-κB and ERK activation (Figures 6D and 6E). Together, these results suggest that Nlrp12-deficient myeloid cells, particularly macropha" @default.
• W2121722333 created "2016-06-24" @default.
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• W2121722333 date "2011-11-01" @default.
• W2121722333 modified "2023-10-18" @default.
• W2121722333 title "The NOD-Like Receptor NLRP12 Attenuates Colon Inflammation and Tumorigenesis" @default.
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